SPG6, a hereditary spastic paraplegia, has insidiously progressive lower-extremity spasticity with degeneration of long central nervous system axons. The SPG6 locus maps to chromosome 15q11.2 and we identified dominant-negative mutations in NIPA1 in two unrelated families. NIPA1 and the adjacent, related NIPA2 gene encode 9-transmembrane (9-TM) domain proteins we hypothesize are transporters. A third, unlinked family member is mutated in a recessive ichthyosis, a skin disease;these studies implicate this 9- TM protein family in one branch of eicosanoid biology. NIPA1 is expressed in neurons and dendrites, in the endoplasmic reticulum (ER) and associated vesicles. In SPG6, we propose that disease results from prevention of a normal NIPA1 function in axonal maintenance and/or from a secondary mechanism involving an unfolded protein response (UPR) and ER trapping of SPG proteins. Preliminary data on cell line expression of EGFP-tagged Nipal and SPG6 supports both hypotheses, since Nipal induces long cellular extensions and the SPG6 mutation induces the UPR. We propose to examine these hypotheses of NIPA1 function and the mechanism by which SPG6 mutations produce spastic paraplegia. Aim 1: To examine our hypothesis that SPG6 mutations generate an UPR and/or otherwise interfere with topology, subcellular localization of NIPA1, or of interactions with other SPG proteins, these parameters will be examined for NIPA1, mutant SPG6 and other wildtype SPG polypeptides in HeLa and neuronal cells. Aim 2: To determine the role of Nipal in normal neurons, a prerequisite to determining if SPG6 mutations interfere with NIPA1 function in vivo, we will (i) induce and characterize HeLa and neuronal cell extensions in tissue culture, (ii) generate a conditional Nipal loss of function mutation in the mouse embryo and postnatally, and (iii) generate developmental zebrafish models by a morpholino antisense approach. Aim 3: To examine the molecular pathological basis for SPG6 mutations in vivo, we will generate spastic paraplegia mouse models by Nipal overexpression using wildtype and SPG6 transgenes, examining similar parameters as for Aims 1 and 2. Aim 4: To test a hypothesis that SPG6 mutations interfere with membrane transport by NIPA1, transport studies will be performed in Xenopus oocytes. Our studies will establish the neuronal roles of NIPA1, the mechanisms by which dominant-negative mutations produce neurological disease, the pathomolecular basis of axonal neurodegeneration in spastic paraplegia, and by identifying NIPA1 transport functions may lead to therapeutic targets in spastic paraplegia and other neurobehavioral diseases. Understanding how neurons develop is critical to therapy for spinal cord injury and disease. This work will use aenetics and biochemistry to identify the function of a aene important for neuron function and disease.